The characterization of cell specific gene expression finds application in a variety of disciplines, such as in the analysis of differential expression between different tissue types, different stages of cellular growth or between normal and diseased states. Fundamental to the characterization of cell specific gene expression is the detection of mRNA. However, the detection of mRNA is often complicated by one or more of the following factors: cell heterogeneity, paucity of material, or limits of low abundance mRNA detection.
One method which has been developed to address at least some of the problems associated with mRNA detection is known as antisense RNA (aRNA) amplification. In this method first strand cDNA is prepared from mRNA using an oligo dT primer that comprises a RNA polymerase promoter region 5' of the oligo dT region. The first strand cDNA is then converted to ds cDNA. Finally, the double stranded (ds) cDNA is contacted with the appropriate RNA polymerase under conditions sufficient to produce aRNA. The method can be adjusted to obtain amplification of the initial mRNA of up to 10.sup.6 fold. The aRNA can then be used in a variety of applications as probe, for cDNA library construction and the like, where such applications include assays for differential gene expression.
Current methods of antisense RNA amplification as described above that employ RNA intermediates are not entirely satisfactory. One potential problem with current methods of antisense RNA amplification is that amplification may be biased (bias refers to the disproportionate amplification of the individual mRNA species in a given population). Another problem is that the amplification products become successively smaller with each succeeding round of amplification. Furthermore, RNA is a labile molecule.
Accordingly, there is interest in the development of improved methods of antisense RNA amplification which do not suffer from one or more the above deficiencies experienced using current methods.